HPLC-ELSD与GC-MS法测定牛乳甘油三酯sn-2位脂肪酸组成

研究建立了快速、准确测定牛乳脂肪甘油三酯sn-2位脂肪酸组成的方法．利用胰脂酶专一水解甘油三酯sn-1和sn-3位置上的脂肪酸得到sn-2单甘油酯和游离脂肪酸,再通过蒸发光散射高效液相色谱分离出sn-2位单甘油酯,然后对其进行衍生,用气相色谱质谱联用仪对sn-2脂肪酸组成进行分析．结果显示用蒸发光散射高效液相色谱法分离sn-2位单甘油酯时方法的回收率达到83．3％～85．1％,该法省去了传统测定中费时费力的薄层色谱分离步骤．用气相色谱质谱联用法对产物进行分析,精密度高,结果可靠．分析结果表明,牛乳脂肪sn-2位脂肪酸由2．57％月桂酸、7．68％豆蔻酸、34．74％棕榈酸、11．56％亚油酸、22．53％油酸和15．21％硬酯酸组成．     

Hydrolyse enzymatique: 1 - approche dans l'etude de l'hydrolyse de diesters: selectivite par competition intermoleculaire

Through competitive experiments, the enzymatic hydrolysis of monoesters has been shown to be influenced by the acyl and alcoxy parts of the ester. Very good selectivities have been observed in some cases with opposite results according to the enzyme involved (horse liver esterase or porcine pancreatic lipase).     

Preparation of a Crosslinked Bioimprinted Lipase for Enrichment of Polyunsaturated Fatty Acids from Fish Processing Waste

Geotrichum sp. lipase modified with a combined method composed of crosslinking and bioimprinting was employed to selectively hydrolyze waste fish oil for enrichment of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in glycerides. Crosslinked polymerization by monomer (polyethylene glycol 400 dimethyl acrylate), crosslinker (trimethylolpropane trimethylacrylate), and photoinitiator (benzoin methyl ether) coupled to bioimprinting using palmitic acid as imprint molecule, resulted in much more effective enzyme preparation used in aqueous hydrolysis reaction. Since the crosslinked polymerization modification maintained bioimprinted property and gave good dispersion of enzyme in reaction mixture, the crosslinked bioimprinted enzyme exhibited higher hydrolysis temperature, enhanced specific activity, shorter hydrolysis time, and better operational stability compared to free lipase. Crude fish oil was treated at 45 °C with this crosslinked bioimprinted lipase for 8 h, and 46% hydrolysis degree resulted in the production of glycerides containing 41% of EPA and DHA (EPA+DHA), achieving 85.7% recovery of initial EPA and DHA. The results suggested that bioimprinted enzymes did not lose their induced property in aqueous environment when prepared according to the described crosslinking–bioimprinting method. It could also be seen that the crosslinked bioimprinted lipase was effective in producing glycerides that contained a higher concentration of polyunsaturated fatty acid with better yield.     

Free Linoleic Acid and Oleic Acid Reduce Fat Digestion and Absorption In Vivo as Potent Pancreatic Lipase Inhibitors Derived from Sesame Meal

Pancreatic lipase catalyzes the cleavage of triacylglycerols at the oil–water interface, and is known as the dominant determiner of dietary fat digestion. Reducing dietary fat digestion and absorption by modulating the activity of pancreatic lipase has become a favorable strategy to tackle obesity. Orlistat is, at present, the only pancreatic lipase inhibitor approved for the treatment of obesity; however, an array of gastrointestinal adverse effects associated with orlistat limits its tolerability. As a safe alternative to orlistat, a number of natural product-derived compounds with varying degrees of pancreatic lipase inhibitory activity have been reported. We herein reported that bioactivity-guided fractionation of sesame meal led to the identification of free linoleic acid and oleic acid as potent inhibitors of porcine pancreatic lipase in vitro with an IC50 of 23.1 µg/mL (82.4 µM) and 11.7 µg/mL (41.4 µM), respectively. In rats, a single oral dose of the mixture of these fatty acids significantly suppressed the elevation of blood triacylglycerol level following fat intake. These results substantiate the role of free linoleic acid and oleic acid as a novel class of natural product-derived functional molecules that act as pancreatic lipase inhibitors, and their potential for healthy, routine-based weight management.     

Rapid access to structured triacylglycerols acylated with n-3 polyunsaturated fatty acids for nutritional applications

In order to better understand the metabolic fate of n-3 polyunsaturated fatty acids (PUFAs), an efficient access to symmetrical and unsymmetrical triacylglycerols (TGs), esterified with PUFAs, with known high purity, is required. In this context, we optimized the esterification of a mixture of glycerols protected as dioxane and dioxolane with PUFAs. The kinetics of this reaction depends on various factors, such as the fatty acid chain length and the stereochemistry of the dioxane. Then, one-pot acetal hydrolysis and esterification of hydroxyl groups led to the desired structured TGs without either double bond isomerization or acyl migration (except when symmetrical TGs are acylated with long-chain saturated fatty acids in external positions). PUFAs location on the glycerol backbone was assayed by NMR, HPLC and pancreatic lipase hydrolysis.     

脂肪酶在有机溶剂中催化酯合成和酯交换反应

报道了猪胰脂肪酶在冻干时的pH值、有机溶剂的极性、反应系统的含水量、温度以及底物碳链的长度和支链对酶在有机相中催化活性的影响规律。利用该酶合成了具有玫瑰香味的月桂酸香茅酯和辛酸香茅酯。     

Contribution of blotting techniques to the study of rapeseeds (Brassica napus L.) lipases

A recent advance in the study of plant lipases involving immunological techniques is presented. In an attempt to characterize lipases of cotyledons from germinating rapeseed seedlings and to investigate an eventual cross-reactivity with animal lipases, we have prepared anti-porcine pancreatic lipase antibodies raised in rabbit. It is shown by enzyme-linked immunosorbent assay and dot-blotting that these antibodies react with lipases in the rapeseed crude extract and in the different cellular fractions obtained by differential centrifugation. Preincubation of the antiserum with the rapeseed crude extract affects the amount of antibodies binding to the porcine pancreatic lipase. We demonstrate immunochemical cross-reactivity between rapeseed and porcine pancreatic lipase. Using the immunoblotting procedure, it is found that antibodies bind specifically to a single polypeptide with a molecular mass of about 55 kDa. Rapeseed lipase activity decreased after immunoprecipitation suggesting that antibodies were bound to some catalytic site residues. We conclude from the data obtained in this study that the two different lipase species present close similarities in amino acid sequence and antigen characteristics.     

薄层色谱-热辅助水解甲基化-气相色谱法测定生物柴油中的甘油酯

建立了薄层色谱-热辅助水解甲基化-气相色谱法测定生物柴油中残余甘油酯含量的方法.样品中的甘油酯经薄层色谱分离,萃取后与三甲基氢氧化硫(0.1 mol/L)各3 μL先后加入到样品杯中,在350℃下,于裂解器中进行衍生化反应,气相色谱测定生成的脂肪酸甲酯,确定甘油酯的含量.生物柴油中常见的甘油一酯、二酯、三酯在60～20...     

Enzymatic hydrolysis of anchovy oil: Production of glycerides enriched in polyunsaturated fatty acids

In an attempt to produce the polyunsaturated fatty acid (PUFA)enriched glycerides, commercially available Turkish anchovy oil (PUFA content of 27%), was hydrolyzed with 1,3-specificRhizomucor miehei lipase. After the hydrolysis, the triglyceride (TG), diglyceride (DG), monoglyceride (MG), and free fatty acid (FFA) composition of the reaction mixture was determined, and fatty acid components of these fractions were analyzed.R. miehei lipase released PUFA extremely slowly, resulting in their accumulation in the TG and DG fractions, especially in TG. The PUFA content in the glyceride mixture (including TG, DG, and MG) increased as hydrolysis progressed. The effects of operational parameters (pH, temperature, time, and enzyme concentration) on the extent of hydrolysis were investigated. Based on these results, optimal reaction conditions were established. At optimal conditions (pH 4.0, 35°C, 3 h, and enzyme concentration of 500 U/g oil), the level of PUFA in the glyceride mixture was raised to 40%. The individual TG and DG fractions contained 45 and 30% PUFA, respectively. Less than 2% of the total PUFA was lost in the FFA fraction.     

Quantitative determination of lipase activity by liquid chromatography-mass spectrometry

We developed a novel in vitro lipase assay based on the quantitation of fatty acids by liquid chromatography-mass spectrometry. Oleic acids enzymatically released from triolein substrates were isolated from the reaction mixture by reverse-phase chromatography, ionized in negative mode electrospray mass spectrometry and quantitated with the aid of [(13)C]-oleic acid internal standard. The enzymatic activity was measured by monitoring oleic acid productions at multiple time points. This method overcomes the substrate and pH limitations of conventional techniques and thus serves as a generic lipase activity assay.     

Expedient Synthesis of (R)-Patulolide A

An efficient derivation of the title compound has been formulated from easily accessible 10-undecenoic acid (1). Thus, dodec-11-en-2-ol (3), prepared from 1, was pyranylated and subjected to bromination with NBS followed by acetolysis to furnish (2E)-1-acetoxy-11-(tetrahydropyranyloxy)dodec-2-ene (5). Its hydrolysis, oxidation, and depyranylation afforded the (2E)-hydroxy ester (9). This, on Candida rugosa lipase-catalyzed acetylation, SeO(2) oxidation, hydrolysis, and Yamaguchi macrolactonization, led to (R)-patulolide A (I) with 67.1% ee. The enantiomeric excess was improved to 97% by first resolving the alcohol 3 via porcine pancreatic lipase catalyzed acetylation and converting the corresponding (R)-acetate (13) to I as done above.     

An improved method for sn-2 position analysis of triacylglycerols in edible oils and fats based on immobilised lipase D (Rhizopus delemar)

The use of lipase D (Rhizopus delemar) immobilised on microporous polypropylene as a replacement for the standard pancreatic lipases used in the stereospecific sn-2 position analysis of triacylglycerols from edible oils and fats is studied. Excellent hydrolysis characteristics are obtained in hexane/methanol solvents at reaction temperatures up to 60 degrees C with hydrolysis times of only 10-20 min. The favourable conditions for the hydrolysis reaction allow fats with higher melting points to be analysed and facilitate coupling of the hydrolysis reaction to the later steps in the analytical protocol. The performance of the new method is compared to that of the standard method using pancreatic lipase. The novel procedure is faster, manual sample handling is reduced, while the results obtained with both methods are comparable. The influence of alkyl-chain length on hydrolysis rates seems to be negligible for the most common vegetable fatty acids. Acyl migration was found to be absent. The short-term repeatability of the method ranges from 10% for fatty acids present at levels close to the detection limits to less than 1% for the major fatty acids. The detection limit is approximately 0.05%. Although the application of the immobilised enzyme in fully automated sn-2 position analysis seems to be promising, the attempts to do this using a packed bed reactor were not successful due to a rapid loss of enzyme activity.     

Improving Stability and Activity of Cross-linked Enzyme Aggregates Based on Polyethylenimine in Hydrolysis of Fish Oil for Enrichment of Polyunsaturated Fatty Acids

Cross-linking of enzyme aggregates from recombinant Geotrichum sp. lipase based on polyethylenimine (PEI) was applied to hydrolyze fish oil for enrichment of polyunsaturated fatty acids successfully. Through acetone precipitation and cross-linking of physical aggregates using glutaraldehyde in the presence of PEI, firmly cross-linked enzyme aggregates (PEI-CLEAs) were prepared. They could maintain more than 65% of relative hydrolysis degree after incubation in the range of 50–55 °C for 4 h and maintain more than 85% of relative hydrolysis degree after being treated by acetone, tert-butyl alcohol and octane for 4 h. PEI-CLEAs increased hydrolysis degree to 42% from 12% by free lipase. After five batch reactions, PEI-CLEAs still maintained 72% of relative hydrolysis degree. Hydrolysis of fish oil by PEI-CLEAs produced glycerides containing concentrated EPA and DHA in good yield. PEI-CLEAs had advantages over general CLEAs and free lipase in initial reaction rate, hydrolysis degree, thermostability, organic solvent tolerance and reusability.     

An Efficient Method for Determination of the Degree of Substitution of Cellulose Esters of Long Chain Aliphatic Acids

The determination of the degree of substitution (DS) of fatty acid cellulose esters, representing a broad range of substituents (C₆, C₁₂, C₁₈ and C₂₂), was performed by alkaline hydrolysis of the ester groups and the quantification of fatty acids by gas chromatography-mass spectrometry (GC-MS) as their trimethylsilyl derivatives. The method was optimized and compared with established techniques for the DS determination (elemental analysis and alkaline hydrolysis/titrimetry). The results demonstrated that alkaline hydrolysis/GC-MS is a rapid, reliable and powerful method for analysis of fatty acid cellulose esters, particularly when different acyl substituents are present.     

Optically active cyclopropanols from the enzymatic resolution of dimethyl α-alkylsuccinates. Synthesis of chiral 2-vinylcyclobutanones and cyclohexenones.

(+)-(R) [or (-)-(S)] dimethyl α-methylsuccinates, obtained by the enantioselective hydrolysis of the racemic diester by porcine pancreatic lipase, undergo acyloin cyclization followed by stereoselective ring contraction to provide 1-alkenylcyclopropanols with high enantiomeric excesses.     

Phospholipase C from Bacillus cereus. Action on some artificial lecithins.

The hydrolysis by phospholipase C from B. cereus of several lecithins of different fatty acyl chain length was examined. The enzyme showed significant activity towards mono-molecularly dispersed short chain lecithins and the reaction obeyed normal Michaelis-Menten kinetics. Rate vs. substrate concentration curves obtained with dihexanoyl-, diheptanoyl- and dioctanoyllecithins showed marked discontinuities in the region of the known critical micelle concentrations for these substrates and distinctly higher rates were obtained just above these levels. Using these three lecithins at levels below their respective critical micelle concentrations, rate increases were noted if the reactions were allowed to proceed to a sufficiently great extent. The presence of deoxycholate in the reaction system had little or no effect on the rate of enzyme-catalysed hydrolysis of lecithins of fatty acyl chain length less than or equal to Cbeta, but for fatty acyl chain lengths greater than C10, significant rate increases occurred. The pH profile for the enzyme activity was also examined.     

Acylglycerols (=Glycerides) from the Glandular Trichome Exudate on the Leaves of Paulownia tomentosa

Chemical investigations of the glandular trichome exudates on the leaves of Paulownia tomentosa (Scrophulariaceae) led to the identification of the thirty acylglycerols (=glycerides) 1 – 30 , including five known ones ( 2, 3, 6, 9 , and 15 ) (Fig. 1). Spectroscopic analysis combined with GC/MS studies of the glycerides and the liberated fatty acids, in the form of trimethylsilyl ether derivatives and trimethylsilylated methyl esters, respectively, established that the constituents belonged to 1,3‐di‐O‐acetyl‐2‐O‐(fatty acyl)glycerols, 1‐O‐acetyl‐2‐O‐(fatty acyl)‐sn‐glycerols, and 2‐O‐(fatty acyl)glycerols, wherein the fatty acyl moiety was either an eicosanoyl or an octadecanoyl group bearing OH and/or AcO groups at the 3‐, 3,6‐, 3,7‐, 3,8‐, or 3,9‐positions. The 1‐O‐acetyl‐2‐O‐[(3R,6S)‐3‐(acetyloxy)‐6‐hydroxyeicosanoyl]‐sn‐glycerol ( 12 ; 20% of the total glycerides), 2‐O‐[(3R,8R)‐3,8‐bis(acetyloxy)eicosanoyl]glycerol ( 17 ; 14%), 2‐O‐[(3R,9R)‐3,9‐bis(acetyloxy)eicosanoyl]glycerol ( 18 ; 12%), and 2‐O‐[(3R)‐3‐(acetyloxy)eicosanoyl]glycerol ( 10 ; 12%) were relatively abundant constituents. The configurations of the stereogenic centers of the fatty acyl moieties were determined by ¹H‐NMR analysis of the monoesters obtained from (R)‐ and (S)‐2‐(naphthalen‐2‐yl)‐2‐methoxyacetic acid ((R)‐ and (S)‐2NMA? OH and the hydroxy‐substituted fatty acid methyl esters (Fig. 2). The configuration at C(2) of the glycerol moiety of the 1‐O‐acetyl‐2‐O‐(fatty acyl)glycerols was determined to be (2S) by chemical conversion of, e.g., G‐2 (= 2 / 3 1 : 10) to (+)‐3‐O‐[tert‐butyl)diphenylsilyl]‐sn glycerol of known absolute configuration.     

Structure-antibacterial activity relationship of secondary metabolites from Ajuga pseudoiva Rob. leaves

A selection of steroids, glycerides, clerodane diterpenoids, and a beta-hydroxy fatty acid methyl ester, all previously isolated from Ajuga pseudoiva leaves, were tested for their antibacterial activity toward three Gram- rods and one Gram+ coccus using the dilution method; MIC values were determined. The results suggested some importance for a free beta-hydroxy group in the fatty acid ester and also in the glycerides and clerodane derivatives; the absolute configurations of the latter, notably at C2, had little influence on activity.     

Thermolysin catalyses the synthesis of cyclodextrin esters in DMSO

Fatty acid esters of cyclodextrins (CDs) were synthesised in a one-step reaction with native CDs as acyl acceptors and vinyl-activated fatty acid esters as acyl donors. Immobilised preparations of thermolysin, subtilisin, the alkaline protease AL-89 and Candida antarctica lipase B were investigated for their catalytic properties regarding transesterification in solvents of increasing hydrophilicity. The synthesis of cyclodextrin fatty acid esters was proved to be catalysed enzymatically by thermolysin in DMSO. The obtained products were analysed by TLC and their structures characterised by NMR, MS and FTIR spectroscopy. With vinyl decanoate as acyl donor β-CD was esterified at all seven glucose C-2 positions resulting in heptakis(2-O-decanoyl)-β-cyclodextrin as the major product. With vinyl butyrate, substitution occurred at all the C-2 and partially at the C-3 or C-6 positions resulting in an average degree of substitution of nine. Between 20% and 25% (w/w) of the acyl donor was converted to esters in 20 h corresponding to an estimated total conversion of the acyl acceptor in the case of maltosyl-β-CD. In the subtilisin and AL-89 catalysed reactions, product formation was simultaneously catalysed non-enzymatically by inorganic buffer salts in aprotic, hydrophilic solvents and with the lipase no products were formed in any of the solvents investigated.     

Synthesis and kinetic resolution of furyl substituted secondary carbinols by porcine pancreatic lipase under solvent free conditions

Chiral furyl substituted carbinols were prepared from the related carbonyl compounds and their enzymatic kinetic resolution was studied by using porcine pancreatic lipase (PPL) in transesterification reaction under solvent free condition. This study revealed that carbinols having one less bulky group on the chiral center are good substrates for PPL and the resolution proceeds with higher ee,s and less reaction time.